Analysis of the Mechanical Characteristics of Human Pancreas through Indentation: Preliminary In Vitro Results on Surgical Samples.

Pancreatic surgery is extremely challenging and demands an extended learning curve to be executed with a low incidence of post-operative complications. The soft consistency of the human pancreas poses a primary challenge for pancreatic surgeons. This study aimed to analyze the preliminary mechanical characteristics of the human pancreas to develop a realistic synthetic phantom for surgical simulations in the near future. Pancreatic specimens, comprehensive of the pancreatic parenchyma and main pancreatic duct, were collected during pancreatic resections and analyzed through nano-bio-indentation (BioindenterTM UNHT3 Bio, Anton Paar GmbH, Graz, Austria) to measure the elastic modulus. Comparisons were made between slow and fast loading rates, immediate and post-freezing analyses, and multipoint indentations. The results demonstrated that a slow loading rate (30 µN/min), immediate analysis, and multipoint measurements are crucial for obtaining accurate values of the elastic modulus of the human pancreas (1.40 ± 0.47 kPa). In particular, the study revealed that analysis after freezing could impact the outcomes of the indentation. Moreover, the study suggested that both the pancreatic parenchyma and the main pancreatic duct should be analyzed to achieve a more precise and comprehensive definition of the. mechanical features of the pancreas. These preliminary findings represent the initial steps toward defining the consistency and mechanical characteristics of human pancreatic tissue with the goal of creating a realistic synthetic phantom.

Design of fluorinated stealth poly(ε-caprolactone) nanocarriers.

The covalent functionalization of polymers with fluorinated moieties represents a promising strategy for the development of multimodal systems. Moreover, polymer fluorination often endows the resulting nanocarriers with improved colloidal stability in the biological environment. In this work, we developed fluorinated pegylated (PEG) biodegradable poly(ε-caprolactone) (PCL) drug nanocarriers showing both high colloidal stability and stealth properties, as well as being (19F)-Nuclear Magnetic Resonance (NMR) detectable. The optimized nanocarriers were obtained mixing a PEG-PCL block copolymer with a nonafluoro-functionalized PCL polymer. The role of PEGylation and fluorination on self-assembly and colloidal behavior of the obtained nanoparticles (NPs) was investigated, as well as their respective role on stealth properties and colloidal stability. To prove the feasibility of the developed NPs as potential 19F NMR detectable drug delivery systems, a hydrophobic drug was successfully encapsulated, and the maintenance of the relevant 19F NMR properties evaluated. Drug-loaded fluorinated NPs still retained a sharp and intense 19F NMR signal and good relaxivity parameters (i.e., T1 and T2 relaxation times) in water, which were not impaired by drug encapsulation.

Intracellular delivery of therapeutic proteins. New advancements and future directions.

Achieving the full potential of therapeutic proteins to access and target intracellular receptors will have enormous benefits in advancing human health and fighting disease. Existing strategies for intracellular protein delivery, such as chemical modification and nanocarrier-based protein delivery approaches, have shown promise but with limited efficiency and safety concerns. The development of more effective and versatile delivery tools is crucial for the safe and effective use of protein drugs. Nanosystems that can trigger endocytosis and endosomal disruption, or directly deliver proteins into the cytosol, are essential for successful therapeutic effects. This article aims to provide a brief overview of the current methods for intracellular protein delivery to mammalian cells, highlighting current challenges, new developments, and future research opportunities.

Design of Functional Pluronic-Based Precursors for Tailoring Hydrogel Thermoresponsiveness and Cell-Adhesive Properties.

In this study, functional Pluronic F127 precursors were designed and synthesized for the preparation of thermosensitive hydrogels. Using linear Pluronic thioacetate and Pluronic multi-acrylate precursors, F127-based hydrogels were prepared through thioacetate deprotection-mediated Michael-type addition. The properties of these gels were compared to those obtained through free radical crosslinking of F127 diacrylate. Temperature was found to have a clear influence on gel swelling as a result of F127 thermoresponsiveness. The macromolecular architecture and functionality of the precursors were also optimized and characterized in terms of gelation kinetics and drug diffusion. In vitro tests were conducted on fibroblasts and endothelial cells to assess their response to cellular adhesion with Pluronic gels that were functionalized with an RGD peptide or pretreated with serum proteins to promote cell adhesion. This study provides a method for creating tailored hydrogels suitable for various biomedical applications, such as soft-tissue engineering, cell encapsulation, wound healing, and sustained delivery of therapeutic molecules.

Analytical Challenges in Diabetes Management: Towards Glycated Albumin Point-of-Care Detection.

Diabetes mellitus is a worldwide-spread chronic metabolic disease that occurs when the pancreas fails to produce enough insulin levels or when the body fails to effectively use the secreted pancreatic insulin, eventually resulting in hyperglycemia. Systematic glycemic control is the only procedure at our disposal to prevent diabetes long-term complications such as cardiovascular disorders, kidney diseases, nephropathy, neuropathy, and retinopathy. Glycated albumin (GA) has recently gained more and more attention as a control biomarker thanks to its shorter lifespan and wider reliability compared to glycated hemoglobin (HbA1c), currently the "gold standard" for diabetes screening and monitoring in clinics. Various techniques such as ion exchange, liquid or affinity-based chromatography and immunoassay can be employed to accurately measure GA levels in serum samples; nevertheless, due to the cost of the lab equipment and complexity of the procedures, these methods are not commonly available at clinical sites and are not suitable to home monitoring. The present review describes the most up-to-date advances in the field of glycemic control biomarkers, exploring in particular the GA with a special focus on the recent experimental analysis techniques, using enzymatic and affinity methods. Finally, analysis steps and fundamental reading technologies are integrated into a processing pipeline, paving the way for future point-of-care testing (POCT). In this view, we highlight how this setup might be employed outside a laboratory environment to reduce the time from measurement to clinical decision, and to provide diabetic patients with a brand-new set of tools for glycemic self-monitoring.

Non-spherical Polymeric Nanocarriers for Therapeutics: The Effect of Shape on Biological Systems and Drug Delivery Properties.

This review aims to highlight the importance of particle shape in the design of polymeric nanocarriers for drug delivery systems, along with their size, surface chemistry, density, and rigidity. Current manufacturing methods used to obtain non-spherical polymeric nanocarriers such as filomicelles or nanoworms, nanorods and nanodisks, are firstly described. Then, their interactions with biological barriers are presented, including how shape affects nanoparticle clearance, their biodistribution and targeting. Finally, their drug delivery properties and their therapeutic efficacy, both in vitro and in vivo, are discussed and compared with the characteristics of their spherical counterparts.

Fluorinated PLGA Nanoparticles for Enhanced Drug Encapsulation and 19 F†NMR Detection.

In the continuous search for multimodal systems with combined diagnostic and therapeutic functions, several efforts have been made to develop multifunctional drug delivery systems. In this work, through a covalent approach, a new class of fluorinated poly(lactic-co-glycolic acid) co-polymers (F-PLGA) were designed that contain an increasing number of magnetically equivalent fluorine atoms. In particular, two novel compounds, F3 -PLGA and F9 -PLGA, were synthesized and their chemical structure and thermal stability were analyzed by solution NMR, DSC, and TGA. The obtained F-PLGA compounds were proven to form in aqueous solution colloidal stable nanoparticles (NPs) displaying a strong 19 F NMR signal. The fluorinated NPs also showed an enhanced ability to load hydrophobic drugs containing fluorine atoms compared to analogous pristine PLGA NPs. Preliminary in vitro studies showed high cell viability and the NP ability to intracellularly deliver and release a functioning drug.

Nanosized Delivery Systems for Therapeutic Proteins: Clinically Validated Technologies and Advanced Development Strategies.

The impact of protein therapeutics in healthcare is steadily increasing, due to advancements in the field of biotechnology and a deeper understanding of several pathologies. However, their safety and efficacy are often limited by instability, short half-life and immunogenicity. Nanodelivery systems are currently being investigated for overcoming these limitations and include covalent attachment of biocompatible polymers (PEG and other synthetic or naturally derived macromolecules) as well as protein nanoencapsulation in colloidal systems (liposomes and other lipid or polymeric nanocarriers). Such strategies have the potential to develop next-generation protein therapeutics. Herein, we review recent research progresses on these nanodelivery approaches, as well as future directions and challenges.

Assessment of increased glomerular permeability associated with recurrent focal segmental glomerulosclerosis using an in vitro model of the glomerular filtration barrier.

The presence of circulating permeability factors (cPFs) has been hypothesized to be associated with recurrence of focal segmental glomerulosclerosis (rFSGS) in renal allografts. The available methods to detect cPFs are complex, not easily repeatable and inappropriate to represent the anatomical characteristics of the three-layer glomerular filtration barrier (GFB). Here we describe a novel method which measures the permeability to bovine serum albumin (BSA) through a three-layer device (3LD). The 3 layers comprise: (1) conditionally immortalized human podocytes (HCiPodo), (2) collagen type IV coated porous membrane and (3) human glomerular endothelial cells (HCiGEnC). Using this method, we found that sera from all rFSGS patients increased albumin permeability, while sera from non recurrent (nrFSGS) and genetic (gFSGS) forms of FSGS did not. The mechanisms underlying the increase of albumin permeability are probably due to endothelial cell damage as an initial event, which was demonstrated by the decrease of Platelet endothelial cell adhesion molecule (PECAM-1 or CD31), while the podocytes' expressions of synaptopodin and podocin were normal. Furthermore, we also found that the plasmapheretic treatment (PPT) eliminated the effect of increasing BSA permeability in sera from rFSGS patients. These preliminary data suggest that our in vitro GFB model could not only be useful in predicting the recurrence of FSGS after renal transplantation (RTx), but also be a valuable in vitro model to study podocyte and endothelial cell biology.

Mannosylated brush copolymers based on poly(ethylene glycol) and poly(ε-caprolactone) as multivalent lectin-binding nanomaterials.

A class of linear and four-arm mannosylated brush copolymers based on poly(ethylene glycol) and poly(ε-caprolactone) is presented here. The synthesis through ring-opening and atom transfer radical polymerizations provided high control over molecular weight and functionality. A post-polymerization azide-alkyne cycloaddition allowed for the formation of glycopolymers with different mannose valencies (1, 2, 4, and 8). In aqueous media, these macromolecules formed nanoparticles that were able to bind lectins, as investigated by concanavalin A binding assay. The results indicate that carbohydrate-lectin interactions can be tuned by the macromolecular architecture and functionality, hence the importance of these macromolecular properties in the design of targeted anti-pathogenic nanomaterials.

Complex Polymeric Architectures Self-Assembling in Unimolecular Micelles: Preparation, Characterization and Drug Nanoencapsulation.

Unimolecular polymeric micelles are a class of single-molecule amphiphilic core-shell polymeric architectures, where the hydrophobic core is well stabilized by the hydrophilic shell, avoiding intermolecular core-core interactions. Multi-arm copolymers with a dendritic core, as well as hyperbranched and comb-like polymers, can form unimolecular micelles easily. In this review, examples of polymers able to form detectable unimolecular micelles will be presented, summarizing the analytical techniques used to characterize the unimolecular micelles and discriminate them from other supramolecular aggregates, such as multi-micelle aggregates. Unimolecular micelles are suitable for the nanoencapsulation of guest molecules. Compared to traditional supramolecular micelles, unimolecular micelles do not disassemble under dilution and are stable to environmental modifications. Recent examples of their application as drug delivery systems, endowed with increased stability and transport properties, will be discussed.

Chitosan/ß-glycerophosphate-based microparticles manufactured by laminar jet break-up technology.

This study is about the use of ß-glycerophosphate (ßGP) to modulate the production of chitosan microparticles through a technology of jet break-up. ßGP has been described as capable of producing chitosan gels without additional complexing agents via a thermal transition (inverse gelation). A preliminary assessment on the effect of temperature on the viscosity and gelation of chitosan/ßGP precursors demonstrated that the crosslinking process was too slow to afford microparticle production via jet break-up. Instead, ßGP was used as a solubilizer to provide stable chitosan solution at neutral pH, which allowed the preparation of microparticles through polyelectrolyte complexation (with triphosphate) under physiological conditions, as opposed to the more conventional method of chitosan solubilisation in acids. Here, the key parameters of the microencapsulation process have been optimized, aiming to produce spherical particle of well-defined size and circularity, as well as toroidal microparticles, with a physico-chemical evaluation of the products.

Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy.

Fetal growth restriction (FGR) in pregnancy is commonly caused by impaired uteroplacental blood flow. Vasodilators enhance uteroplacental perfusion and fetal growth in humans and animal models; however, detrimental maternal and fetal side effects have been reported. We hypothesised that targeted uteroplacental delivery of a vasodilator would enhance drug efficacy and reduce the risks associated with drug administration in pregnancy. Phage screening identified novel peptides that selectively accumulated in the uteroplacental vasculature of pregnant mice. Following intravenous injection, the synthetic peptide CNKGLRNK selectively bound to the endothelium of the uterine spiral arteries and placental labyrinth in vivo; CNKGLRNK-decorated liposomes also selectively bound to these regions. The nitric oxide donor 2-[[4-[(nitrooxy)methyl]benzoyl]thio]-benzoic acid methyl ester (SE175) induced significant relaxation of mouse uterine arteries and human placental arteries in vitro; thus, SE175 was encapsulated into these targeted liposomes and administered to healthy pregnant C57BL/6J mice or endothelial nitric oxide synthase knockout (eNOS-/-) mice, which exhibit impaired uteroplacental blood flow and FGR. Liposomes containing SE175 (0.44mg/kg) or PBS were administered on embryonic (E) days 11.5, 13.5, 15.5 and 17.5; fetal and placental weights were recorded at term and compared to mice injected with free PBS or SE175. Targeted uteroplacental delivery of SE175 had no effect on fetal weight in C57BL/6J mice, but significantly increased fetal weight and mean spiral artery diameter, and decreased placental weight, indicative of improved placental efficiency, in eNOS-/- mice; free SE175 had no effect on fetal weight or spiral artery diameter. Targeted, but not free SE175 also significantly reduced placental expression of 4-hydroxynonenal, cyclooxygenase-1 and cyclooxygenase-2, indicating a reduction in placental oxidative stress. These data suggest that exploiting vascular targeting peptides to selectively deliver SE175 to the uteroplacental vasculature may represent a novel treatment for FGR resulting from impaired uteroplacental perfusion.

The effect of thermosensitive liposomal formulations on loading and release of high molecular weight biomolecules.

Thermosensitive liposomes are clinically-relevant nanocarriers which have been used to deliver chemotherapeutic agents to tumors in combination with local hyperthermia. However, the encapsulation and release of macromolecular therapeutic agents (proteins, nucleic acids, bioactive polymers) is often hindered by their instability during the liposome formation as well as by the low encapsulation efficiency. The objective of this study was to investigate the influence of the thermosensitive liposomal formulation on the encapsulation and release of low and high molecular weight hydrophilic drugs, in order to identify the key parameters to control during nanocarrier design, depending on the specific drug delivery application. Thermosensitive liposomes with different formulations were prepared through the combinations of different lipids, including dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), cholesterol (Chol), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (P-Lyso-PC), and the PEGylated lipid distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000). The thin film hydration method was used for liposome preparation and loading of different water soluble molecules. The encapsulation efficiency and release profiles were investigated for a low molecular weight compound such as carboxyfluorescein (CF), proteins (albumin), and hydrophilic polymers which do not interact with the lipid bilayer, such as a linear dextran and a poly(ethylene glycol)-based star polymer. An optimised liposomal formulation [DPPC/P-lyso-PC/DSPE-PEG2000 90/10/4 (mol/mol) (LTSL)] was chosen for further application in encapsulating therapeutic proteins, such as lysozyme and the brain-derived neurotrophic factor (BDNF), which are recognized as drug carriers and potential therapeutic agents for kidney diseases and neurological disorders.

Ultrasmall polymeric nanocarriers for drug delivery to podocytes in kidney glomerulus.

We explored the use of new drug-loaded nanocarriers and their targeted delivery to the kidney glomerulus and in particular to podocytes, in order to overcome the failure of current therapeutic regimens in patients with proteinuric (i.e. abnormal amount of proteins in the urine) diseases. Podocytes are glomerular cells which are mainly responsible for glomerular filtration and are primarily or secondarily involved in chronic kidney diseases. Therefore, the possibility to utilise a podocyte-targeted drug delivery could represent a major breakthrough in kidney disease research, particularly in terms of dosage reduction and elimination of systemic side effects of current therapies. Four-arm star-shaped polymers, with/without a hydrophobic poly-ε-caprolactone core and a brush-like polyethylene glycol (PEG) hydrophilic shell, were synthesised by controlled/living polymerisation (ROP and ATRP) to allow the formation of stable ultrasmall colloidal nanomaterials of tuneable size (5-30nm), which are able to cross the glomerular filtration barrier (GFB). The effects of these nanomaterials on glomerular cells were evaluated in vitro. Nanomaterial accumulation and permeability in the kidney glomerulus were also assessed in mice under physiological and pathological conditions. Drug (dexamethasone) encapsulation was performed in order to test loading capacity, release kinetics, and podocyte repairing effects. The marked efficacy of these drug-loaded nanocarriers in repairing damaged podocytes may pave the way for developing a cell-targeted administration of new and traditional drugs, increasing efficacy and limiting side effects.

Revisiting Boronate/Diol Complexation as a Double Stimulus-Responsive Bioconjugation.

This study presents a quantitative assessment of the complexation between boronic acids and diols as a reversible and double-stimulus (oxidation and acidification)-responsive bioconjugation reaction. First, by using a competition assay, we have evaluated the equilibrium constants (water, pH 7.4) of 34 boronate/diol pairs, using diols of both aliphatic and aromatic (catechols) nature; in general, catechols were characterized by constants 3 orders of magnitude higher than those of aliphatic diols. Second, we have demonstrated that successful complexation with diols generated in situ via enzymatic reactions, and the boronate complexation was also employed to calculate the Michaelis-Menten parameters for two catechol-producing reactions: the demethylation of 3-methoxytyramine and the 2-hydroxylation of estradiol, respectively, mediated by P4502D6 and P4501A2. Third, we have prepared phenylboronic acid-functionalized hyaluronic acid (HA) and demonstrated the pH and H2O2-responsive character of the adducts that it formed with Alizarin Red S (ARS) used as a model catechol. The versatility and selectivity of the complexation and the mild character of the chemical species involved therefore make the boronate/catechol reaction an interesting candidate for bioconjugation purposes.

Polymer Nanoparticle Engineering for Podocyte Repair: From in Vitro Models to New Nanotherapeutics in Kidney Diseases.

Specific therapeutic targeting of kidney podocytes, the highly differentiated ramified glomerular cells involved in the onset and/or progression of proteinuric diseases, could become the optimal strategy for preventing chronic kidney disease. With this aim, we developed a library of engineered polymeric nanoparticles (NPs) of tuneable size and surface properties and evaluated their interaction with podocytes. NP cytotoxicity, uptake, and cytoskeletal effects on podocytes were first assessed. On the basis of these data, nanodelivery of dexamethasone loaded into selected biocompatible NPs was successful in repairing damaged podocytes. Finally, a three-dimensional in vitro system of co-culture of endothelial cells and podocytes was exploited as a new tool for mimicking the mechanisms of NP interaction with glomerular cells and the repair of the kidney filtration barrier.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.

The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics.

Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies.

Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.